Fig. 3. Characterization of CASAAV-RNF20/40 and RNF20/40-depleted CMs.

a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes (a) was performed to measure protein levels of RNF20, RNF40, H2Bub1, and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 (b) showed significant 74% reduction (P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve (c) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography (d) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts (n = 3 mice) at P7 showed dramatic upregulation of Myh7^YFP compared with controls (n = 3 mice). Scale bars = 200 μm. f–n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26^Cas9-GFP/+;Myh7^YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7^YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h–n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls (h; P < 0.0001), with a moderate increase in length-to-width ratio (i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation (P = 0.0207). k–l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m, n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density (P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: *P < 0.05, **P < 0.001.